The broad aim of this project is to elucidate how cells transport organelles and molecular complexes along microtubules (Mts) to specific destinations. To address this problem, we must explain the molecular basis and the overall logic for how different kinesin motors are mobilized to carry specific cargoes. To his end, we will substantiate our hypothesis that processive kinesin motors, which are primarily soluble in the cell, are in an inhibited ground state until they are bound and activated by their cargoes. How the non-motor regions of kinesins direct motor activity and cargo binding is the research plan's principle concern. We will identify, within the non-motor regions of a few kinesins, the domains required for binding cargo and regulating motor activity. Such domain-mapping studies will bring to light plausible mechanisms for cargo binding and motor regulation, and most importantly, pinpoint candidate-binding sites for proteins that implement these activities. Preliminary studies along these lines on conventional kinesin validate this strategy - our domain-mapping studies indicate that the inhibited state of conventional kinesin depends on kinesin light chain (KLC) and involves a folded conformation, in which the C-terminus of kinesin heavy chain (KHC) interacts with its own motor domain. Moreover, these studies have identified the KLC tandem tetratricopeptide repeats (KLC TPRs) as prime candidates to bind factors that either link kinesin to its cargo or activate kinesin's interaction with Mts. In the first specific aim, we will now isolate these KLC TPR partner proteins, using biochemical and genetic approaches, and characterize their functions. In a similar fashion, the second and third specific aims propose to investigate the non-motor regions of a number of other kinesins (heterotrimeric kinesin II and monomeric KIF1A,B, & C) whose cargoes are known. By identifying, within the non- motor regions of these kinesins, sites involved in cargo binding and motor activation, we will again develop a rationale for isolating the protein factors that implement these activities. It is the identification of these interacting proteins that is this plan's ultimate goal. To ensure that we isolate proteins that are functionally relevant, we will introduce, into the nonmotor regions of these kinesins, mutations that disrupt motor regulation or cargo binding. Such kinesin mutants are the keystone of our proposal - proteins that bind specifically to wild-type, but not mutant, sites are likely to be the bona fide partners in vivo. The generation and use of such negative controls fundamentally distinguishes this proposal from previous attempts to identify proteins that interact with kinesins.